Construction of expression vector of specific two-protein multi-antigen epitope gene of Mycoplasma pneumoniae

Objective To construct the specific two-protein multi-antigen epitope expression vector of Mycoplasma pneumoniae(Mp) for enhancing the sensitivity of the recombinant protein antigen.Method Antigen epitope sequences of Mp adhesion protein P116 was screened by bioinformatics method.P116 protein gene was obtained by PCR point mutations technology and recombined with pMD-T vector and then transformed into E.coli JM109.Recombinant plasmid was identified and analyzed by restriction enzyme mapping and gene sequence.The P116 fragment was purified and recovered by enzyme digestion and then linked with pGEX6P-1-P1 DNA fragment to produce a recombinant DNA which was transformed into E.coli JM109 strain.Recombinant protein was purified with Glutathione Sepharose 4B.Molecular weight and immunogenicity of recombinant protein was analyzed relatively by SDS-PAGE and immunoblotting.Result The P116 gene fragment amplified by PCR was 597 bp.And compared with the known gene sequence in the GenBank,except for the two mutations from the UGG to UAG,the other nucleotide sequence of the gene was 100% matched.SDS-PAGE analysis showed that the molecular weight(Mr) of the multi-epitope recombinant protein was 77.8 kDa.Western blot analysis showed this recombinant protein could react with Mp polyvalent rabbit serum.Conclusion This study successfully constructed the expression vector of two-protein specific multi-antigen epitope of Mp,and the recombinant protein of this vector possesses specific immune response with Mp.The sensitivity of the recombinant protein will be identified and evaluated further.